Research: Attachment the respiratory epithelium is believed to be an early step in the development of pneumonia. Klebsiella produce filamentous organelles called fimbriae which carry adhesive protein subunits called adhesins. This proposal will further characterize the Klebsiella type 3 fimbrial adhesin responsible for adherence to respiratory epithelium. The initial experiments will identify which DNA sequence(s) of the type 3 fimbrial adhesin gene, mrkD, are critical for proper function of the adhesin. The two necessary recombinant plasmids producing transformed bacteria that express nonadhesive type 3 fimbriae (pFK28) and type 3 fimbrial adhesin protein (pFK58) were previously constructed. Hence, in this study, site-specific and random mutations in pFK58 can be produced and the appropriate mutations identified by DNA sequencing. The effect of the mutations on adherence by cotransformed bacteria will be quantified by three established adherence assays to type V collagen, human tracheal cells, and lung tissue sections. In subsequent experiments, type 3 fimbrial adhesin antibodies will be developed to assist in experiments to localize the adhesin on Klebsiella type 3 fimbrial filament as well as on mutant strains, developed in earlier experiments, that may demonstrate increased or decreased adherence. Additional experiments will utilize synthetic nonameric peptides, derived from the region(s) of the mrkD gene shown to be important, to determine the primary amino acid sequence required for adherence by the type 3 adhesin and to determine if these regions encompass epitopes recognized by adhesin-specific antisera. In the last group of experiments the applicant will use a novel method of allelic exchange to generate specific deletions in the Klebsiella mrk gene cluster within the native chromosomal DNA. Subsequently the impact on adherence by these specifically mutated Klebsiella will be assessed in the three adherence assays.